Structure of the dopamine D3 receptor bound to a bitopic agonist reveals a new specificity site in an expanded allosteric pocket

Although aminergic GPCRs are the target for ~25% of approved drugs, developing subtype selective drugs is a major challenge due to the high sequence conservation at their orthosteric binding site. Bitopic ligands are covalently joined orthosteric and allosteric pharmacophores with the potential to boost receptor selectivity, driven by the binding of the secondary pharmacophore to non-conserved regions of the receptor. Although bitopic ligands have great potential to improve current medications by reducing off-target side effects, the lack of structural information on their binding mode impedes rational design. Here we determine the cryo-EM structure of the hD3R coupled to a GO heterotrimer and bound to the D3R selective bitopic agonist FOB02–04A. Structural, functional and computational analyses provide new insights into its binding mode and point to a new TM2-ECL1-TM1 region, which requires the N-terminal ordering of TM1, as a major determinant of subtype selectivity in aminergic GPCRs. This region is underexploited in drug development, expands the established secondary binding pocket in aminergic GPCRs and could potentially be used to design novel and subtype selective drugs.


Introduction
While G protein-coupled receptors (GPCRs) form the largest family of drug targets, accounting for more than a third of FDA-approved drugs 1 , developing subtype-selective drugs is a major challenge.This is especially true for aminergic GPCRs, which include 42 receptors (dopamine, serotonin, adrenaline, histamine, acetylcholine and trace amine receptors) with high sequence similarity.In the most closely related aminergic receptor subtypes, sequence identity often exceeds 80% of the orthosteric binding site (OBS) residues.Such conservation supports neurotransmitter promiscuity 2 between subtypes, but results in undesired off-target side effects of drugs that only bind in the OBS 3 .
Controlling drug selectivity for aminergic receptors has the potential to improve current therapies and it could be achieved by the design of bitopic molecules [4][5][6] .These are ligands generated by covalently joining two pharmacophores, a primary pharmacophore (PP), that usually targets the OBS, and a secondary pharmacophore (SP), that targets an allosteric or secondary binding pocket (SBP) generally divergent in sequence and/or structure within the target receptor [4][5][6][7] .Hence, bitopic molecules have been proposed to have a separate "message-address" system wherein an agonist/antagonist, the message, is linked to a pharmacophore binding to the SBP, which contains the address 5,8 .Indeed, several bitopic compounds with enhanced receptor selectivity have been developed for GPCRs, specially for the muscarinic receptor where a variety of allosteric modulators is available [9][10][11] .Overall, bitopic ligands present a rational approach to develop molecules with enhanced functionality and selectivity, however there is scarce structural information on their binding modes and development relies heavily on classical structureactivity relationships 12 .Here we aim to understand the molecular basis of a selective bitopic drug that distinguishes between two closely related aminergic GPCRs, the human dopamine D2 receptor (D2R) and dopamine D3 receptor (D3R).These receptors share 78% sequence identity at the transmembrane segment and 100% identity at the OBS, making their pharmacological distinction a notoriously hard challenge 13,14 .D2R and D3R differ in brain distribution and signaling properties, and are both targeted by current antipsychotics and drugs for the treatment of neurological diseases (such as Parkinson's disease 15,16 ).
Although agonists with some selectivity exist, treatments are still suboptimal due to a lack of selectivity and different effects originating from the activation of both receptors 17,18 .
Hence, new subtype selective molecules are likely to help understand their physiological role as well as providing leads for improved therapeutics.Furthermore, selective activation of D3R may yield neuroprotective effects in Parkinson´s disease, hence harboring potential in the treatment of neurodegeneration 19,20 .
In this work, we determined the cryo-electron microscopy (cryo-EM) structure of the human D3R bound to a bitopic and full agonist (FOB02-04A) and coupled to a GαOβγ heterotrimer.Together with functional assays, mutagenesis, docking studies, and molecular dynamics simulations, we determine the binding mode and basis for D3R selectivity of this compound.The bitopic molecule occupies the OBS and protrudes towards the outside of the ligand binding pocket to contact a new allosteric site at the extracellular vestibule of D3R formed by TM2-ECL1 and TM1.This region is of high sequence and structural variability and expands the established aminergic SBP, opening new avenues to develop subtype-selective bitopic drugs, potentially across other aminergic GPCRs.

Overall cryo-EM structure of the hD3R:GαOβγ:scFv16 bound to a bitopic ligand
The D3R:GO heterotrimer:FOB02-04A complex was produced by co-expressing the hD3R (L 3.41 W mutation following Ballesteros-Weinstein numbering 21 ), the dominant negative GαO subunit 22 , Gβ1 and Gγ2 in insect cells (see Methods) (Supplementary Fig. 1A).The D3R L3.41W was previously used in structural studies 23 and was validated in this work using cellular BRET assays 24 , where it displayed a virtually identical ligand-induced activation as the wild-type D3R (Supplementary Fig. 1B).The bitopic FOB02-04A was synthesized as previously described 9 and was added before complex solubilization from insect cell membranes.The scFv16 25 (which binds to the GαO:Gβ interface) was incorporated prior to size exclusion chromatography.The structure of the complex was then solved by single-particle cryo-EM (Fig. 1 and Supplementary Fig. 2).Positioning the ligand binding pocket at the center of the cryo-EM box improved the resolution at the D3R extracellular region (Methods and Supplementary Fig. 3), allowing to separate two FOB02-04A and binding site conformations -Conformation A (to a global resolution of 3.05 Å) and B (global resolution of 3.09 Å), which mainly differed in the position of the bitopic SP and residues around the SBP (Fig. 1 and Supplementary Fig. 2).We will initially focus on Conformation A unless otherwise stated since Conformation B was concluded to be a non-productive antagonistic conformation (see below).Both final cryo-EM maps were of sufficient quality to build confidently the D3R, the Gαβγ proteins, the scFv16 and the bitopic FOB02-04A ligand (Supplementary Fig. 4 and Supplementary Table 1).Both D3R conformations were built from residues H29 (Conformation A)/Y32(Conformation B) to C400 with missing residues for intracellular loop 3 (ICL3) (residues I224 to R322, both excluded from the model).No cholesterol (or cholesterol hemisuccinate) or lipid molecules were found around the transmembrane part of the receptor, consistent with previous reported structures of the D2R [26][27][28] and D3R 23,28,29 and in contrast to D1R, D4R and D5R where cholesterol was bound to the transmembrane segment 28,30,31 .

Activation mechanism and GO coupling of the D3R bound to FOB02-04A
The D3R:GαOβγ:FOB02-04A displays the characteristic structure of a GPCR:G protein complex, with resemblance to the previously determined structures of D3R coupled to a Gi heterotrimer 28,29 (e.g.RMSD of 1.036 Å for 1022 Cα for the pramipexole bound structure, PDB 7CMU).No major conformational changes are found in the D3R when comparing it bound to pramipexole (PDB 7CMU), PD128907 (PDB 7CMV), rotigotine (PDB 8IRT) or FOB02-04A (0.535 Å RMSD over 253 Cα in the pramipexole bound as example) aside from the ordering of the extracellular region of TM1 (see below).The D3R activation induced by FOB02-04A follows the canonical conformational changes 32 , i.e. a downward shift of the toggle switch W342 6.48 , a conformational change of the PIF (I118 3.40 , F338 6.44 ), DRY (D127 3.49 , R128 3.50 , Y129 3.51 ) and NPxxY (N379 7.49 , P380 7.50 , P Y383 7.53 ) motifs, which end up with an ~9 Å outward swing of the cytoplasmic end of TM6 and inward movement of TM7 towards the core of the receptor as compared to the inactive state 23 (Supplementary Fig. 5).The coupling of the GO heterotrimer to the D3R occurs through two interfaces: a first major interface located between the GαO C-terminal α5, that engages mainly with the intracellular part of TM3, TM5 and TM6 of the D3R (I344, L348, C351, L353, and Y354 in GaO packing against R128 3.50 , A131 3.53 , V132 3.54 , I211 5.61 , L215 5.65 , R218 5.68 , R222 5.72 , R323 6.29 , K326 6.32 , A327 6.33 and M330 6.36 in D3R) with contributions from TM7 and TM2 (Fig. 2A).Of note, from molecular dynamics (MD) simulations spanning five independent 0.6 µs runs of the D3R bound to FOB02-04A and coupled to GαOβγ within a membrane bilayer, alternating salt bridge interactions occurred between D341 G.H5. 13 (superscript denotes CGN numbering system 33 ) of the GαO C-terminal α5 and the guanidinium groups of R218 5.68 and R222 5.72 in D3R (Supplementary Fig. 6).A second interface is located at the intracellular loop 2 (ICL2), which makes interactions in a pocket formed by the GαO N-terminal helix, the C-terminal α5 and the loops connecting the β-strands.The interaction is also held together by unspecific electrostatic charges between the receptor and the Gα protein conserved among Gi/O coupled receptors 28,29 .
The D3R has been shown to couple preferentially to GO compared to Gi 34 .The current D3R:GO structure allows us to compare it with the previously determined D3R:Gi complex to search for potential differences that could explain such D3R coupling preference (Fig. 2B-C).Overall, both structures exhibit a similar interface area, with D3R-GO having only a slightly lower buried surface area than D3R:Gi (959.4Å 2 and 1051.8Å 2 for GO and Gi coupled D3R respectively).However, a smaller interface area is usually seen in GO vs Gi couplings irrespective of selectivity 35,36 .Additionally, both structures present a similar outward swing in TM6 irrespective of GO or Gi coupling (Fig. 2B-C), in line with previous observations of the same receptor coupled to different Gα proteins keeping the magnitude of TM6 outward swing 37,38 .However, differences occur when looking at the C-terminal a5 interactions of GO vs Gi.In the case of GaO, the terminal Y354 G.H5.26 points towards TM5, in contrast to its equivalent F354 G.H5. 26 in Gai, which is sandwiched between R323 6.29 and K345 G.H5.17 (this residue is specific of Gai, A345 G.H5.17 in GaO).As a result, D3R TM6 is shorter in the GO coupled structure (Fig. 2C).Furthermore, previous studies suggested that native ICL contacts are essential to achieve GO selectivity in D3R 34,39 .Indeed, structural differences were also found at the interaction made by ICL2 where, in GO, Q139 34.54 moves away from the a5 of GO to interact with K32 in the aN.Such interaction was further confirmed in MD simulations, whose interacting distance remained constant along the five trajectories spanning 0.6 µs each (Supplementary Fig. 6).Additionally, the interaction between Q144 ICL2 and E28 in the Gi αN is lost when coupled to GO due to a replacement of E28 G.HN.52 by an isoleucine as well as the slight difference in the positioning of GO with respect to the receptor (Fig. 2D).Overall, these differences might contribute to the D3R GO selectivity.

The binding mode of the bitopic agonist FOB02-04A at D3R
Bitopic molecules are composed of a PP (binding at the OBS), an SP (binding at the allosteric site), and a linker.FOB02-04A is a full agonist bitopic molecule composed of a non-catechol PP (based on PF592,379, an aryl-morpholine-based scaffold), a SP with an indole-amide group, and a (1R,2S)-cyclopropyl linker moiety whose chirality has been optimized for ligand binding and selectivity 9,40,41 (Fig. 3A).The cryo-EM density allowed modelling of the three bitopic components.Unlike other agonists, which target exclusively the bottom of the pocket, FOB02-04A binds to the OBS and runs along a narrow channel towards the allosteric site in the extracellular vestibule, interacting with residues from TM1-3 and TM5-7 (Fig. 3B-D).The SP of FOB02-04A is found protruding out of the tight channel to bind in the extracellular vestibule of D3R, occupying most of the ligand binding pocket, in contrast to pramipexole which only occupies 23% of the pocket volume (Fig. 3B-C).Each component of the bitopic molecule (PP, linker and SP) occupies a different region within the D3R pocket, overall defined by a combination of hydrophobic and polar interactions, as described in Fig. 3E.
The PP pocket at the OBS is defined by strong salt bridge interactions with D110 3.32 , and a cavity formed by S196 5.46 , F345 6.51 , F346 6.52 , W342 6.48 , V111 3.33 , T115 3.37 and I183 ECL2 , with an additional weak H-bond with S192 5.42 (Fig. 3).To correlate structural information with functional activity, most of the residues involved in ligand binding were mutated to alanine, following quantification of their surface expression and measurement of their ligand-induced activation using functional BRET assays in HEK293T cells 24 (see Methods and Supplementary Fig. 7).At the OBS there were critical residues which showed no detectable activity when mutated to alanine such as the conserved D110 3.32 , which forms a stable charge interaction with almost all agonists in aminergic receptors, and W342 6.48 , the conserved toggle switch residue at the bottom of the OBS pocket that is essential for signaling.Additionally, I183 ECL2 , which sandwiches the ligand from the extracellular side (ECL2), V111 3.33 and T115 3.37 had a significant impact on agonist potency when mutated (Fig. 3H-I).V111 3.33 is specifically relevant for FOB02-04A, since its mutation does not have an impact on the D3R-induced activation by pramipexole, rotigotine and PD128907 28,29 .In turn, T115 3.37 is relevant for FOB02-04A and pramipexole in contrast to PD128907 and rotigotine.Finally, an agonist interaction with S192 5.42 is found within most aminergic receptor-agonists pairs, however it seems to be less important for FOB02-04A binding (Fig. 3, Supplementary Fig. 7).This is in line with non-catechol agonists not relying heavily on S192 5.42 for binding and activation 42 (also observed for pramipexole 29 ).A conserved hydrophobic pocket between T369 7.39 and F345 6.51 is efficiently occupied by the rotigotine, pramipexole and PD128907 propylamine group, while it is barely occupied by a methyl group by FOB02-04A (Supplementary Fig. 8).This may explain the lack of effect of F345 6.51 A upon activation by FOB02-04A and suggests that a larger hydrophobic group at this position might improve its binding.
The linker component of the FOB02-04A, which connects PP and SP, interacts with residues at the established SBP in aminergic receptors 3,12 , an unexploited region in pramipexole and PD128907 but occupied by the propylthiophene group in rotigotine 28 .
The pocket is formed by residues V86 2.61 , F106 3.28 , T369 7.39 and Y373 7.43 and has been proposed to have different plasticity among dopamine receptors, and hence a source for ligand specificity 28 .In the case of FOB02-04A, three residues showed a significant reduction in activity when mutated to alanine: Y373 7.43 , F106 3.28 and V86 2.61 .Y373 7. 43 A showed non-detectable activity and, although this residue is known to be relevant for maintaining the D110 3.32 geometry to make the conserved charged interactions with agonists, its mutation does not have such a pronounced effect on the activity of pramipexole, dopamine and PD128907 29 as it has on the activity of rotigotine or FOB02-04A.This suggests a direct role in ligand-induced activation of the bitopic molecule.
Additionally, alanine mutation of F106 3.28 and V86 2.61 showed reduced efficacy.This is likely to be FOB02-04A specific since V86 2.61 A did not reduce efficacy upon pramipexole activation 29 .Overall, the linker connecting the PP and SP has an active role in the D3R selective binding and function of FOB02-04A and its related bitopic analogs 29 .
Finally, the FOB02-04A SP binds in a groove-shaped pocket at the receptor extracellular region, denoted as SBP2-ECL1-1 and formed by the tips of TM1 and TM2, and ECL1.
Remarkably, in contrast to prior D3R structures-whether in active or inactive conformations-the outermost extracellular residues undergo a rearrangement that positions H29 1.32 's imidazole group, situated between TM2 and TM7, to stack with the 1H-indole group of the ligand SP.Given the absence of H29 1.32 in preceding D3R cryo-EM 28,29 and crystal structures 23 , we sought to ascertain the orientation of the imidazole moiety of H29 1.32 .For this purpose we performed comparative MD simulations, involving two D3R complexes coupled to GαOβγ and bound to either FOB02-04A or pramipexole (PDB ID: 7CMU), both within a membrane bilayer and aqueous milieu and executed across five parallel runs of 0.6 µs each.MD analysis elucidated a more consistent localization of H29 1.32 between TM2 and TM7 when complexed with FOB02-04A relative to pramipexole.In this conformation, H29 1.32 side chain is directed towards the SBP2-ECL1-1, engaging with the SP of FOB02-04A bitopic ligand (Supplementary Fig. 9).
Although the protonated N(ε) atom of H29 1.32 imidazole and the carboxyl entity of E90 2.65 are too distant to support strong polar or ionic interactions, the D3R complex with the bitopic ligand FOB02-04A exhibited a narrower distance distribution than in pramipexole complex (Supplementary Fig. 9).In addition, in the D3R-FOB02-04A complex, the N(ɛ) atom of H29 1.32 consistently interacts with the backbone oxygen of E90 2.65 .Conversely, when complexed with pramipexole, three of the five trajectories show this distance consistently surpassing 10Å.This observation reinforces that, while in the FOB02-04A:D3R complex the H29 1.32 side chain is predominantly positioned in the SBP2-ECL1-1 where it is stabilized by the ligand, in the pramipexole-bound complex H29 1.32 side chain points away, likely due to the absence of the allosteric pharmacophore in pramipexole (Supplementary Fig. 9).
To gain further insights into the SBP2-ECL1-1 role, we mutated all residues within this pocket to alanine (except for G94 ECL1 which was deleted) and measured ligand-induced activation using BRET2 assays.These experiments revealed that deletion of G94 ECL1 , which prevents ECL1 from reaching the SP is essential for FOB02-04A activity (Fig. 3H-I).A previous study identified G94 ECL1 as a key determinant for binding of a similar bitopic molecule, however, only a reduction in affinity was observed (using radioactive ligands and fluorescence) 43 while, in the current study, ligand-induced activity seemed to be fully ablated.This suggests that FOB02-04A could potentially still bind in the DG94 ECL1 variant (although with lower affinity) but triggers no detectable Gabg activation, hence G94 ECL1 is likely to determine affinity and efficacy.Further mutational analysis of residues within SBP2-ECL1-1 identified L89 2.64 and H29 1.32 as key residues, with only a slight reduction in potency (~3-fold and ~6-fold reduction in EC50 for H29 1.32 and L89 2.64 respectively) but a significant decrease in efficacy upon alanine mutation (especially for H29 1.32 ) (Fig. 3I).Previous studies predicted how slight variations in the position of the PP at the D3R OBS could modulate compound efficacy 44 .Since several residues at the SBP modulate FOB02-04A efficacy, it is likely that the linker and SP conformation are currently optimal to position the PP for maximal efficacy at the OBS, and that mutations around the SBP restrict conformations of the FOB02-04A PP to less efficacious alternatives.This scheme yields a marked segregation of the functional roles of the protein residues for each bitopic component.While mutations significantly decreasing potency (>100-fold the EC50) are primarily found at the OBS, mutations at the SBP mainly decrease FOB02-04A efficacy (Fig. 3G).This suggests that the SP is not only involved in D3R selectivity (see below) but also in optimally positioning the PP for activity.Such conclusions are in line with previous suggestions originating in computational and functional assays 45 .Interestingly, additional controls where activation of the H29 1.32 A variant was tested with pramipexole, an agonist that does not reach H29 1.32 , also displayed a reduction in efficacy (data not shown).Hence, we could not quantify the contribution of this residue on bitopic binding since it readily displayed an effect on the intrinsic receptor efficacy.It is not unusual for residues at the most extracellular sites to have an impact in intrinsic receptor function 46 .
Additional analysis of the MD trajectories with the D3R-FOB02-04A complex suggested a more robust interaction of FOB02-04A with D3R than pramipexole.This was observed by looking at the stable salt bridge interaction between the trans-cyclopropyl amine group of FOB02-04A and the carboxyl group of D110 3.32 in D3R (which underscores the stable binding pose of the 6-(aminopyridin-3-yl)-5-methylmorpholine PP moiety) (Supplementary Fig. 6).However, for the pramipexole-bound D3R complex, three out of five MD trajectories displayed substantial deviations in either the equivalent salt bridge interaction with pramipexole amino group, as well as the interactions distance between S196 5.46 in D3R and the pramipexole's amino group.Since pramipexole and FOB02-04A have similar binding affinities, the propensity of pramipexole towards dissociation observed during MD simulations suggests potential faster association and dissociation rates, in line with the larger bitopic molecule requiring longer times for association and dissociation (Supplementary Fig. 6).
Overall, the bitopic agonist FOB02-04A uses all three components (PP, linker and SP) to make critical interactions with the ligand binding pocket, since each component contributes with one critical interaction which, if mutated, the ligand-induced activation as a whole, is eliminated.This highlights that selective bitopic molecules are required to bind en bloc and that the SP which contains the address component is required to contribute significantly to the overall ligand function, otherwise selectivity would be lost.

Structural basis of FOB02-04A D3R/D2R selectivity
The bitopic FOB02-04A ligand has been designed for its PP to carry the agonist message while the SP carries the address, and has been reported to be 50-fold more selective for D3R over D2R 9 .Since quantification of selectivity at the D3R/D2R is assay and conditiondependent 6,9,13 , we measured the D3R/D2R selectivity using cellular BRET assays, which confirmed the 50-fold selectivity (Supplementary Fig. 7).D3R and D2R have 78% sequence similarity at the transmembrane region and, residues within interacting distance of FOB02-04A, showed high structural similarity and 100% sequence identity at the OBS and established SBP 24 .However, FOB02-04A interactions with the G94 ECL1 and H29 1.32 within the SBP2-ECL1-1 form a region that is structurally and sequence diverse between D3R/D2R.The D3R TM2-ECL1 harbors an extra glycine residue that is absent in D2R (93GGV95 in D3R vs 98GE99 in D2R), which allows this region to interact with the SP in the D3R and not in the D2R (Fig. 4A).Deletion of the extra glycine G94 ECL1 in D3R ablates ligand induced activation by FOB02-04A (Fig. 3I and Supplementary Fig. 7).This is in line with previous studies where similar bitopic molecules showed reduced affinity in D3R lacking G94 ECL1 43 .This reduction in activity makes G94 the most critical residue for D3R/D2R selectivity.Additionally, H29 1.32 is positioned in TM1, the most sequence diverse transmembrane helix in GPCRs and that, within D3R/D2R, shows both sequence and structural diversity (Fig. 4A).Further exploiting this unforeseen H29 1.32 has the potential to contribute with selectivity at the functional level.
Selectivity can arise from sequence diversity, structural divergence as well as differences in structural plasticity.Using sequence alignments and the recent explosion in GPCR structural information we assessed whether the SBP2-ECL1-1 is a site of high diversity that could be exploited to develop subtype selective drugs in other aminergic GPCRs.The analysis showed that the SBP2-ECL1-1 is variable either in sequence, structure or both within most aminergic receptor subtypes (Fig. 4B-F).The amount of diversity at the SBP2-ECL1-1 varies within each subfamily, with the least variable being the muscarinic receptors where TM1 is too far apart to contribute in all available structures and the equivalent G94 position is only different in M3R (N131 ECL1 vs a glycine residue in M1, M2, M4 and M5).
However, there are marked differences in several other subgroups.First, the serotonin 5-HT1 and 5-HT2 groups show variable sequence or structure at the G94 ECL1 equivalent position while TM1 is too far apart (Fig. 4C and D).Additionally, the recent structural determination of all five dopamine receptors (D1R-D5R) highlighted the SBP2-ECL1-1 as the most variable region between them 28 .Finally, there are groups with marked differences at the SBP2-ECL1-1 site, e.g. the ARa2A-2C subgroup.ARa2A-2C show differences at the G94 ECL1 equivalent position, while they have an increasingly ordered TM1 which could potentially contribute with specific interaction in each receptor.While in ARa2B TM1 is far apart, it is longer in ARa2A where it could contribute with main chain atoms of Y43 and in ADa2C where the N-terminus folds over the TM2-ECL1 site providing with additional specific residues (Fig. 4D).In ARb1-3, the TM2-ECL1 has structural and sequence divergence that could be used to design highly subtype selective bitopic molecules (Fig. 4C).Overall, the SBP2-ECL1-1 site is a major specificity region that is underexploited for developing subtype selective drugs.However, this site is far away from the canonical ligand binding site and might be better accessible with bitopic molecules.

Alternative FOB02-04A conformation at the ligand binding site
Docking of FOB02-04A to the D3R reliably reproduced its binding mode when compared to the cryo-EM structure.Yet, a second conformation of FOB02-04A was revealed with comparable docking scores, suggesting a second plausible orientation (Fig. 5C).In the alternative binding mode, termed Conformation B, the 1H-indole-2-carboxamide SP of FOB02-04A is seen to interact with a less hydrophobic pocket defined by the polar side chains S182 ECL2 , Y365 7.35 , as well as V360 ECL3 and P362 7.32 residues, termed hereafter SBPECL2-ECL3.Notably, π-π stacking interactions between the 1H-indole part of FOB02-04A and Y365 7.35 stabilizes Conformation B (Fig 5).MD simulations indicated that the indole SP of FOB02-04A oscillates between SBP2-ECL1-1 (Conformation A) and the comparatively less hydrophobic SBPECL2-ECL3 (Conformation B).A detailed examination of the proximity between D3R E90 2.65 and the FOB02-04A SP (accentuated with a red palette) juxtaposed with proximity measurements between D3R Y365 7.35 and the FOB02-04A SP (illustrated in green pallet) provides insights into the temporal predominance of

FOB02-04A's Conformation A versus Conformation B (Fig 5D). Subsequent frequency
analyses showed that Conformation A, that engages SBP2-ECL1-1, is predominant with an estimated 80% prevalence, in contrast to the 20% observed for Conformation B, targeting SBPECL2-ECL3 region (Figure 5D and Supplementary Fig 6).This information triggered a targeted search for Conformation B within the cryo-EM dataset, which resulted in a model at 3.09 Å resolution (Fig. 1, Supplementary Fig. 2 and 4).In this model, cryo-EM density supports the second conformation for the FOB02-04A SP so as to make π-π stacking interactions with Y365 7.35 in a similar manner as found in docking and MD simulations (Fig. 5A-B).Interestingly, in this cryo-EM map, the extracellular residues of TM1, including H29 1.32 , are not resolved, reminiscent of the pramipexole, rotigotine and PD128907 bound D3R structures (Fig. 5B).This suggests that binding of the SP to the SBP2-ECL1-1 stabilizes the TM1 conformation described above (in agreement with our MD simulations).A comparison of particle numbers between cryo-EM models of Conformation A and B also supported a predominance of Conformation A over B (~60%).With such relative abundance both conformations are expected to contribute to function, however, mutating Y365 7.35 to alanine (affecting only Conformation B) did not have an impact while the DG94 ECL1 variant (affecting only Conformation A) fully ablated Gabg dissociation.Such functional outcome does not respond to two functional conformations in equilibrium, but rather we propose that this second conformation is acting as an antagonist or weak partial agonist.Such hypothesis would be in line with our previous observation that residues at the SBP as well as the position of the SP are highly relevant for an optimal positioning and efficacy of the PP.In support of this, a minor twist of the PP at the OBS is observed in Conformation B with respect to Conformation A, and minor modifications at the position of the ligand at the D3R OBS have been shown to regulate ligand efficacy.However, we cannot rule out that the slight difference in PP position is a consequence of the low map resolution.If Conformation B was truly an antagonist, and these two conformations are in equilibrium, mutation of Y365 7.35 should drive all the ligand into the first conformation A, and an increase in potency should result (since Conformation B acts like an antagonist).Indeed, a slight increase in potency was observed in the Y365 7.35 A (Fig. 3 and Supplementary Fig. 7), but it was not statistically significant.However, both conformations have similar relative abundance, therefore a major impact in activity is not expected.We then assessed whether Conformation B could be occurring at D2R, since this residue is conserved in D2R and could account for the binding affinity of FOB02-04A at D2R.However, Y408 7.35 A in D2R did not result in a signaling loss in functional assays (Supplementary Fig 7).Since D2R is more plastic than D3R, the binding mode of this bitopic molecule to D2R might be hard to predict and additional studies would be required.Overall, this second conformation of the bitopic molecule highlights that large flexible molecules might adopt alternative non-productive conformations that might hinder progress in drug development if non-detected.

Discussion
Aminergic receptors are highly relevant drug targets, but the high sequence and structural similarity within the family poses a great challenge to developing subtype-selective drugs.
Here, we have reported the cryo-EM structure of the human D3R in complex with the D3R-selective bitopic agonist, FOB02-04A, and coupled to a GO heterotrimer.FOB02-04A binds D3R with all three components (PP, linker and SP), fully exploiting the OBS, established SBP and a new extended SBP2-ECL1-1 that confers FOB02-04A with D3R selectivity.This SBP2-ECL1-1 is structurally and/or sequence diverse also in aminergic receptors and could potentially be used to develop subtype-selective ligands.Especially interesting is the TM1 contribution to ligand binding since it is the most sequence diverse transmembrane region in GPCRs, rarely contributes to ligand binding, and could be exploited through the use of bitopic molecules with the required composition and length.
Mutational profiling of the ligand binding site showed a marked segregation in functional roles of the residues at the OBS and the SBP.While the majority of mutations that impaired potency were located mainly at the OBS, mutations that impaired efficacy were enriched at the SBP.This highlights the relevant role of the SP binding in optimally positioning the PP at the OBS for maximal activity.The computational design of bitopic molecules might benefit from taking such roles into consideration.Additionally, the mutational analysis pointed to a mutually PP, linker and SP-dependent binding mode, i.e. all components contribute with essential interactions for the en bloc binding of the bitopic molecule.This is likely required when higher selectivity is desired since independent binding might yield promiscuous PP binding.Therefore, the message and address components should not be treated as separate entities when developing specific bitopic molecules, but rather working together in tandem with the appropriate linker in between 6 .
A second antagonistic conformation of the FOB02-04A bitopic molecule is proposed which suggests that care should be taken when developing subtype selective bitopic molecules, since the position of the PP at the OBS seems to be altered easily (at least for the D3R in the case of FOB02-04A) and bitopic molecules tend to be large and flexible, and alternative non-productive conformations might obscure highly specific and potent conformations in functional assays.Such problems likely contribute to the challenges associated with developing agonistic bitopic molecules 6 .Including structural determination in the drug development pipeline is likely to accelerate future progress.
Additional structural information on other bitopic-receptor complexes might shed light on this topic.
Regarding the D3R/D2R selectivity, a recent report describing the structures of the five dopamine receptors (D1R-D5R) pointed towards H 6.55 as a specificity determinant, since this residue changes conformation between D2-like receptors in an agonist-dependent manner 28 .While H 6.55 was located far away from the bitopic molecule under study, molecules with combined interactions at H 6.55 and the new extended SBP2-ECL1-1 site have the potential to yield highly specific molecules within D2-like receptors.Such molecules could help to improve current treatments targeting the D3R, a current target for Parkinson´s disease and other neurological disorders and neuropsychiatric disorders, including substance use disorders [47][48][49] .
Overall, this work extends the usable SBP in aminergic receptors exploiting an extracellular region of high sequence and structural variability and highlights new insights and pitfalls into the development of highly selective subtype selective bitopic molecules with desired functional efficacies.

Construct design and molecular cloning
subjected to CTF refinement and Bayesian polishing following a 3D classification focused on the receptor (with a mask around the receptor) that yielded 429,908 particles.
Refinement of this set of particles yielded a model at 3.16 Å but poor cryo-EM density at the SBP.To improve map quality at the ligand binding site two parallel processing paths were pursued with the 429,908 particle set: 1) a recentering of the particles at the ligand binding site (re-extracted in a 320-pixel box) followed by 3D classification (resulting in 360,038 particles), and 2) 3D classifications with a mask at the extracellular half of the receptor followed by a recentering of the particles at the ligand binding site (as described before) which were further 3D classified (resulting in 176,315 particles).The two sets of particles were merged and duplicates removed, yielding 275,383 particles which were refined using for the last iteration a mask that precluded the GαO-helical domain and the micelle.Post-processing resulted in a cryo-EM map for Conformation A at 3.05Å.
Conformation B was obtained by performing a 3D classification on the 429,908 particle set with a mask on the extracellular half of the receptor resulting in a model with 252,959 particles which were subsequently re-centered at the ligand binding site and further 3D classified.Particles belonging to best model, with 201,219 particles, were subjected to heterogeneous refinement and 159,184 particles were lastly refined through non-uniform refinement in CryoSPARC 56 .This resulted in a cryo-EM map at 3.09 Å according to the gold-standard FSC of 0.143.Local resolution was calculated using CryoSPARC for both models.

Model Building
Model building and refinement was carried out using the CCP-EM software 57 and Phenix 58 .The D3R, Gβ1, Gγ2 and scFv16 starting coordinates were taken from the Gαicoupled D3R structure (PDB code 7CMV) 29 .The GαO starting coordinates were taken from the GαO-coupled α2β adrenoreceptor structure (PDB code 6K41).D3R was modelled from residue H29 to I223 and from R323 to C400 in conformation A (conformation B starts at Y32).GαO was modelled from T4 to K54, T182 to V234 and N242 to Y354.
Jelly-body refinement was performed in REFMAC5 59 followed by manual modification and restraint real space refinement in Coot 60 and Phenix.A dictionary describing the ligand FOB02-04A and its coordinates was created using AceDRG 61 and manually fitted into the density for its latter refinement in real space using Coot and Phenix.B factors were reset to 40 Å 2 prior to refinement.The final model achieved good geometry (Supplementary Table 1) with validation performed in Coot, EMRinger 62 and Molprobity 63 .The goodness of fit of the model to the map was carried out using Phenix, using of a global model-vs-map FSC correlation (Supplementary Table 1).
Cellular BRET assays pEC50 were determined using cellular BRET2 assays with the TRUPATH system 24 .50,000 cells/well were seeded in previously poly-lysined 96-well white plates with clear bottom.The following day, cells were transfected with TransIT-2020 (Mirus Biosciences) at ratio of 2:1:1:1 of D3R:GαOA-RLuc8:Gβ3:Gγ9-GFP2 (7:1:1:1 for D3R Y373A , D3R ΔG94 and D2R Y408A ) following manufacturer instructions.After 48 h, the medium was replaced by 90 µl/well of freshly prepared assay substrate buffer (1×Hank's balanced salt solution, 20 mM HEPES pH 7.4, Coelenterazine 400a 7.5 µM).10µl of each concentration of FOB02-04A was added and the plate was read using a CLARIOstar (BMG Labtech) with 400 nm (RLuc8-Coelenterazine) and 498.5 nm (GFP2) emission filters at integration times of 1.85 s.BRET ratios were calculated as the ratio of GFP2 signal to Rluc8 signal.Data analysis was performed using GraphPad Prism 8.0.1.Data were normalized and a four-parameter logistic curve was fit into the data.Data are presented as mean ± SEM of three independent experiments performed in technical triplicate.

Surface expression quantification
HEK293T cells were plated in previously poly-lysined 96-well white plates (50,000 cells/well) and transfected the next day with the D3R and D2R variants using PEI MAX® at a 2:1 ratio (PEI:DNA).After 48 h, cells were washed twice with 1X Phosphate Buffered Saline (PBS) and fixed with 4% paraformaldehyde for 20 minutes at RT. Cells were then washed three times with PBS for 5 minutes and 100 µl of 1X PBS with 5% BSA (w/v) was added to each well and incubated at RT for 30 minutes.Subsequently, media was replaced with 1X PBS-5% BSA with an anti-Flag HRP conjugate (1:10,000) and incubated at RT for 30 minutes.Cells were then rinsed twice with PBS and 50 µl of HRP substrate (Clarity Max™ Western ECL Substrate) was added to each well and incubated for 5 minutes prior to chemiluminescence detection using a CLARIOstar (BMG Labtech).
Data analysis was performed using GraphPad Prism 8.0.1 Chemiluminescence values were normalized to D3R WT and presented as a ratio of D3R WT.Data are presented as mean ± SEM of three independent experiments performed in technical triplicate.optimization of the side chain residues.Prior to conducting molecular docking, pramipexole underwent chiral definition and formal charge assignment.The compounds' molecular models were created from their two-dimensional representations, and their three-dimensional geometry was refined using the MMFF-94 force field 74 .For docking simulations, a biased probability Monte Carlo (BPMC) optimization approach was employed, adjusting the internal coordinates of the compound based on pre-calculated grid energy potentials of the receptor 75 .The grid potentials, while preserving the receptor's conformational state, considered receptor flexibility through the utilization of "soft" van der Waals potentials.All-atom docking was performed with the energyminimized structure of FOB02-04A employing an effort value of 5.The ligand docking box was selected to encompass the extracellular half of the protein for potential grid docking.At least 10 independent docking runs with 3 conformations in each were conducted, starting from random conformations.Consistency among the docking results was determined by comparing ligand conformations from the best ten docking runs.The unbiased docking procedure did not rely on distance restraints or any predefined information regarding the ligand-receptor interactions.From these docking experiments, two top-scoring docking solutions, referred to as conformation A and conformation B, representing FOB02-04A bound to D3R complexes, were further refined.This refinement involved successive rounds of minimization and Monte Carlo sampling, focusing on the ligand conformation and including sidechain residues within 5 Å of the binding site.All the above-mentioned molecular modeling operations were performed in the ICM-Pro v3.9-2b molecular modeling and drug discovery suite (Molsoft LLC) 67 .